The homeostasis of hematopoietic stem cells (HSCs) is regulated by intricate signaling pathways and interactions with the microenvironment or stem cell niche. One important signaling axis for HSC biology is thrombopoietin (TPO) and its receptor Mpl, signaling through Janus Kinase (JAK2). TPO-/- or Mpl-/- mice show diminished HSC self-renewal. In contrast, mice lacking the adaptor protein Lnk harbor a >10-fold increase in HSC numbers and superior engraftment. We discovered that Lnk regulation of HSC self-renewal partially depends on Mpl. In support of this genetic interaction, we found that Lnk directly binds to phosphorylated tyrosines in JAK2, and TPO-mediated activation of JAK2 is potentiated in Lnk-/- HSCs. Furthermore, Lnk-/- progenitor cells (HPCs) home more efficiently than wild type (WT) controls, and Lnk-/- mice exhibit a marked expansion in extramedullary distribution, indicating Lnk affects HSC interaction with the niche. Consistently, we found that Lnk deficiency enhances HPC adhesion to fibronectin through 21 integrins. We thus hypothesize that Lnk negatively restricts stem cell pool by controlling HSC cell cycle kinetics and survival through Mpl/JAK2, and regulates HSC interaction with stem cell niches by modulating integrin signaling. Specific Aim 1: To investigate the role of Lnk in controlling HSC cell cycle kinetics, survival, and exhaustion. We aim to investigate how the expanded stem cell pool is achieved in Lnk-/- BM, by examining HSC cell cycle kinetics, survival and quiescence status of HSCs during development and after stress. In addition, we will study HSC exhaustion affected by Lnk deficiency using serial BMTs. Specific Aim 2: To investigate the role of Lnk in controlling HSC homing, adhesion, and interaction with HSC niches. We will extend our primary studies on progenitor cells to ask if Lnk deficiency enhances HSC homing/adhesion using BMT assays. In dissecting the mechanisms of Lnk-dependent HSC homing/adhesion, we will focus on two known major pathways: 21 integrin and stromal-derived factor-1 (SDF-1). Furthermore, we will test whether Lnk affects integrin signaling in HSCs through TPO/Mpl. Specific Aim 3: To investigate how Lnk coordinates HSC self-renewal, proliferation/survival, homing, and interaction with the niche. The ostensibly distinct HSC properties are inter-related. We will investigate how perturbation of integrin-initiated signaling will affect WT and Lnk-/- HSC/PC proliferation, survival, and engraftment. Furthermore, we will study the role of Lnk in HSC homing and division kinetics in relationship to micro-anatomic localization in vivo using two-photon/confocal microscopy on live animals upon transplanted. Specific Aim 4: To test Lnk-JAK2 interaction in regulating HSC functions in vivo. Our genetic data suggest Lnk functions in part through its direst association with JAK2 to control HSC homeostasis. Here we will test this model in vivo by generating two Knockin mice that bear point mutations in Lnk and JAK2 to disrupt their interaction. We believe our studies will advance our understanding of the mechanisms underlying stem cell homeostasis and facilitate clinical applications to stem cell therapy. PUBLIC HEALTH RELEVANCE: In mammals, hematopoietic stem cells (HSCs) are responsible for the daily production of billions of mature blood cells throughout adult life, and abnormal stem cell homeostatic mechanisms are associated with a variety of human disorders including aplastic anemia and leukemia. The research proposed here aims to achieve a better understanding of how an intrinsic signaling molecule, Lnk, regulates stem cell expansion, homing, and engraftment. We believe our studies will advance not only our understanding of the mechanisms underlying normal stem cell regulation, but also provide new insights into abnormal stem cell signaling in various lymphomas and leukemias, and facilitate clinical applications to stem cell therapy.